GB Virus C (GBV-C) is a flavivirus that is closely related to hepatitis C virus; however, it does not appear to replicate in the hepatocyte or cause disease in humans. Nevertheless, it is a common infection in humans, with approx. 1.8 percent of healthy blood donors persistently infected, and up to 40 percent of immunocompromised people infected. We and several other groups have shown that HIV-infected people who are co-infected with GBV-C have delayed mortality compared to HI V-infected individuals without GBV-C infection. We also developed an infectious molecular clone of GBV-C, and demonstrated that it replicates in the CD4+ T cell subset of cultured primary peripheral blood mononuclear cells (PBMC?s). Co-infection of PBMC?s with GBV-C and HIV demonstrate inhibition of HIV replication, even when GBV-C infection was initiated after HIV infection was already established, thus the inhibition occurs at a step in HIV replication following HIV attachment and entry GBV-C infection did not cause a decrease in cell viability or metabolic activity, thus the inhibition is not due to toxicity to CD4+ T cells. GBV-C replication in PBMC?s is slow and inefficient, and thus is a difficult model system for studying HIV-GBV-C interactions. In this proposal, we propose to 1)better characterize GBV-C replication in PBMC?s and viral mutations that occur during adaptation to growth in cell culture, 2) develop GBV-C infection models in cultured cell lines and/or isolated T cell cultures, 3) thoroughly investigate GBV-C and HIV replication in vitro, specifically focusing on the effects of GBV-C replication on cellular gene expression, and 4) create recombinant GBV-C viruses expressing heterologous peptides and RNA sequences for use as potential therapeutic agents. Since GBV-C does not appear to cause human disease, causes persistent infection of humans, and appears to replicate in the same cell population as HIV, it has potential to target HIV-specific inhibitors to HIV-infected cells. These studies will better define the mechanism by which GBV-C infection interferes with HIV replication, and may allow the development of novel therapeutic approaches for controlling HIV disease.